High risk human papillomaviruses (HPVs) are central to the development of cervical cancer and the deregulated expression of high risk HPV oncogenes is a critical event in this process. Here, we find that the cell protein nucleolin binds in a sequence-specific manner to the HPV18 enhancer. The DNA binding activity of nucleolin is primarily S phase specific, much like the transcription of the E6 and E7 oncoproteins of HPV18 in cervical cancer cells. Antisense inactivation of nucleolin blocks E6 and E7 oncogene transcription and selectively decreases HPV18+ cervical cancer cell growth. Furthermore, nucleolin controls the chromatin structure of the HPV18 enhancer. In contrast, HPV16 oncogene transcription and proliferation rates of HPV16+ SiHa cervical cancer cells are independent of nucleolin activity. Moreover, nucleolin expression is altered in HPV18+ precancerous and cancerous tissue from the cervix uteri. Whereas nucleolin was homogeneously distributed in the nuclei of normal epithelial cells, it showed a speckled nuclear phenotype in HPV18+ carcinomas. Thus, the host cell protein nucleolin is directly linked to HPV18-induced cervical carcinogenesis.
KRAS mutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRAS mutations, and the different methods reported concordant results (kappa values >0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRAS mutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis.
In this study, typing of human papilloma virus (HPV) was performed in squamous cell carcinomas of the lung (n=26) as well as putative primaries of head and neck (n=21) and female genital tract (n=5) of the same patients, to test whether additional information to discriminate lung primaries from metastases can be gained by a direct comparison of the HPV status in both tumors. In 3 (14.2%) patients with head and neck as well as lung squamous cell carcinoma, an identical HPV subtype could be detected in both tumors suggesting metastatic disease. In 9 (42.9%) cases, discordant HPV status strongly suggested secondary primaries of the lung. In the remaining 9 (42.9%) patients, no HPV was evident in either tumor. In all patients with carcinomas of the cervix uteri an identical HPV subtype was detected in the cervical and in the lung tumor. In conclusion, the results suggest HPV typing, a method routinely used in cervical biopsies for years, as a very useful diagnostic tool to discriminate primary from metastatic squamous cell carcinoma of the lung, which in our cohort in 57.1% of cases allowed for almost definite classification.
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